DsdX is the second D-serine transporter in uropathogenic Escherichia coli clinical isolate CFT073

Abstract

d-Serine is an amino acid present in mammalian urine that is inhibitory to Escherichia coli strains lacking a functional dsdA gene. Counterintuitively, a dsdA strain of E. coli clinical isolate CFT073 hypercolonizes the bladder and kidneys of mice relative to wild type during a coinfection in the murine model of urinary tract infection. We are interested in the mechanisms for uptake of d-serine in CFT073. d-Serine enters E. coli K-12 via CycA, the d-alanine transporter and d-cycloserine sensitivity locus. CFT073 cycA can grow on minimal medium with d-serine as a sole carbon source. The dsdX gene of the dsdCXA locus is a likely candidate for an additional d-serine transporter based on its predicted amino acid sequence similarity to gluconate transporters. In minimal medium, CFT073 dsdX can grow on d-serine as a sole carbon source; however, CFT073 dsdX cycA cannot. Additionally, CFT073 dsdXA cycA is not sensitive to inhibitory concentrations of d-serine during growth on glycerol and d-serine minimal medium. d-[(14)C]serine uptake experiments with CFT073 dsdX cycA harboring dsdX or cycA recombinant plasmids confirm that d-serine is able to enter E. coli cells via CycA or DsdX. In whole-cell d-[(14)C]serine uptake experiments, DsdX has an apparent K(m) of 58.75 microM and a V(max) of 75.96 nmol/min/mg, and CycA has an apparent K(m) of 82.40 microM and a V(max) of 58.90 nmol/min/mg. Only d-threonine marginally inhibits DsdX-mediated d-serine transport, whereas d-alanine, glycine, and d-cycloserine inhibit CycA-mediated d-serine transport. DsdX or CycA is sufficient to transport physiological quantities of d-serine, but DsdX is a d-serine-specific permease.

FIG. 1.

Time-dependent d-serine uptake. Samples were incubated with 0.1 mM d-[¹⁴C]serine (5.5 Ci/mol) for the indicated times. Uptake was stopped with addition of an excess of buffer, and samples were filtered and measured.

FIG. 2.

Kinetic evaluation of d-serine transport. Samples were treated with the indicated concentration of d-[¹⁴C]serine for 20 s, then the reaction was stopped with an addition of excess buffer, and the sample was filtered and measured. DsdX had an apparent K_m of 58.75 μM and an apparent V_max of 75.96 nmol · mg⁻¹ · min⁻¹. CycA had an apparent K_m of 82.40 μM and an apparent V_max of 58.90 nmol · mg⁻¹ · min⁻¹.

FIG. 3.

Substrate analysis. Samples were preincubated with the indicated amino acid for 3 min, at which time d-[¹⁴C]serine was added and the mixture was allowed to incubate for an additional 3 min. Data are plotted as the percentage of wild-type uptake. Percent wild-type uptake of ∼100, no effect; <100, inhibitor; >100, enhancer of transport. (a) The dsdX-complemented strain; (b) the cycA-complemented strain.

FIG. 4.

The d-cycloserine effect requires a H⁺ gradient. Samples were pretreated as indicated with 60 mM d-cycloserine and/or 10 mM CCCP for 3 minutes. Samples were then exposed to d-[¹⁴C]serine for an additional 3 min. The ratio of signal with pretreatment to signal with no pretreatment is plotted.

FIG. 5.

Working model of the dsdCXA locus and d-serine metabolism. Both DsdX and CycA are predicted to lie in the inner membrane (5), and our observations are in agreement with this prediction. cycA transcription is controlled by σ⁵⁴ and Nac (27, 40). Once d-serine enters the cell, it interacts with DsdC, which then induces dsdXA expression. d-Serine is degraded to pyruvate and ammonia by DsdA.